Biomarker for detecting cancer

ABSTRACT

Methods for detecting colorectal cancer in a subject by detection of a galactose-containing 40 kDa molecule in a serum sample from the subject. Methods for quantifying the amount of a galactose-containing molecule in a serum sample are also provided.

This application claims the benefit of U.S. Provisional PatentApplication No. 61/545,675, filed Oct. 11, 2011, the entirety of whichis incorporated herein by reference.

The invention was made with government support under Grant Nos.RO1CA69480 and UO1CA68400 awarded by the National Institutes of Health.The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of molecularbiology, immunology and oncology. More particularly, it concerns methodsfor detecting biomarkers linked to the development of cancer.

2. Description of Related Art

Modern medicine has developed an arsenal of therapies that can bebrought to bear against cancer. However, a major factor in theeffectiveness of such therapies is the stage of the cancer beingtreated. Late stage cancers and cancer cells that have alreadymetastasized from their site of origin remain difficult to treat andcontinue to result in high rates of mortality among patients. Hencesuccessful anti-cancer therapy is largely dependent upon early,accurate, diagnosis. Many cancer diagnostic techniques, however, areinaccurate or invasive, reducing the opportunity for early detection andsuccessful treatment. For example, in the case of colorectal cancer theprimary diagnostic technique, colonoscopy, is viewed as highly invasiveand typically only applied every 5-10 years and only after the age of50. Thus, there remains a need for accurate and non-invasive techniquesfor cancer detection.

SUMMARY OF THE INVENTION

In a first embodiment there is provided a method for detecting acolorectal cancer, comprising (a) obtaining a serum sample of a subject;(b) desialylating the serum; (c) contacting the desialylated serum withan antibody that binds haptoglobin or a cancer-associated haptoglobinglycoform to form a complex, wherein the complex comprises the antibodyand a galactose-containing molecule; and (d) detecting thegalactose-containing molecule on the complex with a detectable lectinthat binds galactose, wherein an elevated level of thegalactose-containing molecule detected as compared with a control levelis indicative of a colorectal cancer. In certain aspects, a method ofthe embodiments is defined as an in vitro method.

In a further embodiment there is provided a method for detecting acolorectal cancer, comprising (a) obtaining a serum sample of a subject;(b) desialylating the serum; (c) contacting the desialylated serum withan antibody that binds haptoglobin to form a complex, wherein thecomplex comprises the antibody and a galactose-containing molecule; (d)detecting the galactose-containing molecule on the complex with adetectable lectin that binds galactose; and (e) determining the presenceof (or the probability of the presence of) a colorectal cancer based onan elevated level of the galactose-containing molecule detected ascompared with a control level. In certain aspects, a method of theembodiments further comprises performing or obtaining the results of oneor more secondary tests to determine the presence of a colorectalcancer. For example, the secondary test can be, without limitation, afecal occult blood test (FOBT), a colonoscopy or a secondary blood test(e.g., a test for levels of carcinoembryonic antigen (CEA) in theblood).

In still a further embodiment there is provided a method for quantifyinga galactose-containing molecule in a serum sample comprising (a)obtaining a serum sample of a subject; (b) desialylating serum; (c)contacting the desialylated serum with an antibody that bindshaptoglobin to form a complex, wherein the complex comprises theantibody and a galactose-containing molecule; and (d) detectinggalactose on the complex with a detectable lectin that binds galactose,thereby quantifying the galactose-containing molecule in the sample.

In certain aspects of the embodiments, step (b) further comprisesdiluting the serum between about 25-fold and 150,000-fold, 100-fold and100,000-fold, 1,000-fold and 50,000-fold, or 10,000-fold and 50,000-fold(e.g., at least about 20-fold, 25-fold, 100-fold, 500-fold, 1,000-fold,5,000-fold, 10,000-fold or 15,000-fold) before desialylating the dilutedserum. As used herein “diluting” means mixing of a sample with a largervolume of a second non-sample solution (e.g., water). As used herein“fold dilution” refers to the dilution factor as compared to a serumsample that has not been mixed with any additional solution (i.e., anunadulterated serum sample).

In a further embodiment there is provided a method for detecting acolorectal cancer, comprising (a) obtaining a serum sample of a subjectwherein the serum sample has been diluted between 25-fold and150,000-fold; (b) desialylating the diluted serum; (c) contacting thedesialylated serum with an antibody that binds haptoglobin to form acomplex, wherein the complex comprises the antibody and agalactose-containing molecule; and (d) detecting galactose-containingmolecule on the complex with a detectable lectin that binds galactose,wherein the elevated level of the galactose-containing molecule detectedas compared with a control level is indicative of a colorectal cancer.

In still a further embodiment there is provided a method for quantifyinga galactose-containing molecule in a serum sample comprising (a)obtaining a serum sample of a subject wherein the serum sample has beendiluted between 25-fold and 150,000-fold; (b) desialylating the dilutedserum; (c) contacting the desialylated serum with an antibody that bindshaptoglobin to form a complex, wherein the complex comprises theantibody and a galactose-containing molecule; and (d) detectinggalactose-containing molecule on the complex with a detectable lectinthat binds galactose, thereby quantifying the galactose-containingmolecule in the sample.

Thus, certain aspects a method is provided for quanifying a galatosecontain molecule in a serum sample in accordance with the foregoingembodiments wherein an elevated level of the galactose-containingmolecule detected as compared with a control level is indicates that thesubject has an elevated risk of colorectal cancer. Thus, in someaspects, a method of detecting colorectal cancer is provided comprisingadministering at least a second test for colorectal cancer to a subjectidentified as having a risk for colorectal cancer. For example, thesecond test can be a blood test, stool test of a colonoscopy.

Thus, in some aspects, obtaining a serum sample comprises obtaining adiluted serum sample. For example, a serum sample can be a sample thathas been diluted by at least about 30-fold, 40-fold, 50-fold, 60-fold,70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold, 10,000-foldor 15,000-fold.

Certain aspects of the embodiments concern obtaining a serum sample of apatient. The serum sample can, for example, be directly obtained bydrawing blood from a patient. In certain cases the sample is obtainedfrom a third party (e.g., a doctor or clinic) or is a frozen bankedsample.

In certain aspects methods of the embodiments concern desialylating adiluted serum sample. For example, the sample subjected to desialylationcan be a sample that has been diluted by about or at least about25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 500-fold, 1,000-fold, 10,000-fold or 15,000-fold prior todesialylation. A diluted sample can be desialylated by a variety ofmethods, such as by treatment with an enzyme or a mild acid. Forexample, a diluted serum sample can be treated with a neuraminidaseenzyme (e.g., a Vibrio cholerae neuraminidase). In certain aspects, adiluted sample is treated with a mild acid such as H₂SO₄, and optionallyheating the sample. For example, a diluted serum sample can be treatedwith 0.5 NH₂SO₄, and heated to about or at most about 50° C., 60° C.,70° C., 80° C. for a period of about or at least about 30 min, 60 min or90 min.

Certain aspects of the embodiments involve contacting a desialylated anddiluted serum with an antibody. For example, in some aspects the sampleis diluted to between 25-fold and 150,000-fold, 100-fold and100,000-fold, 1,000-fold and 100,000-fold, 10,000-fold and 100,000-foldor 15,000-fold and 150,000-fold (e.g., about a 20.000-fold dilution)before contacting the serum with an antibody. Thus, in certain aspects asample is diluted before desialylation, after desialyation or both.

Antibodies for use according to the embodiments include, withoutlimitation, polyclonal and monoclonal antibodies that bind tohaptoglobin (e.g., human haptoglobin). For example, the antibody can bean antibody that was raised against a purified human haptoglobin, suchas a rabbit polyclonal antisera (e.g., the H-8636 antisera availablefrom Sigma). An anti-haptoglobin antibody, can in some cases, be labeledor immobilized. For example, in some aspects, the antibody isimmobilized on a bead (e.g., a magnetic bead) or a surface (e.g., in awell of a plate).

Some aspects of the embodiments involve a step of washing a complexcomprising the antibody and a galactose-containing molecule with a washsolution (i.e., contacting the complex with a volume of wash solution).For example, washing a complex comprising the antibody and agalactose-containing molecule can comprise washing the complex 1, 2, 3,4 or more times with a solution comprising a physiological amount ofsalt and a physiological pH, such as PBS. Optionally a wash solution cancomprise a detergent such as TWEEN-20 (e.g., about 0.01% to about 0.1%TWEEN-20).

In further aspects, methods of the embodiments concern detecting agalactose-containing molecule on a complex (i.e., a complex comprisingan antibody and a galactose-containing molecule) with a detectablelectin that binds galactose. For example, detecting agalactose-containing molecule can comprise (i) contacting the complexwith a lectin that binds galactose and (ii) detecting the bound lectin.In certain aspects, the method further comprises (i) contacting thecomplex with a lectin that binds galactose, (ii) washing the complex 1,2, 3, 4 or more times with a wash solution; and (iii) detecting thebound lectin. Examples of lectins for use according to the embodimentsinclude, without limitation mammalian galectin-3, Ricinus communislectin, Datura stramonium lectin, Erythrina cristagalli lectin, orLycopersicon esculentum lectin. In some aspects, the lectin is labeledwith a detectable label. In still further aspects, the lectin can bedetected with a labeled lectin-binding moiety (e.g., a labeledlectin-binding antibody).

The skilled artisan will recognize that the methods for detecting alectin will depend on the type of label that is employed. For example,in some aspects, a lection comprises an affinity label (e.g., biotin)and the label is itself detected by the binding of a further moleculeslinked to a reporter (e.g., avidin linked to a reporter). In furtheraspects, a lectin may itself be labeled with a reporter. Reportermolecules for use according to the embodiments include, withoutlimitation, dyes, fluorophores, radionuclides and enzymes. For example,in certain aspects, the reporter is a enzyme, such a peroxidase, thatamplifies the detection signal by virtue of its catalytic activity.

Some aspects of the embodiments concern detecting a colorectal cancer bydetecting an increased level of a galactose-containing molecule. Incertain aspects, detecting an increased level of thegalactose-containing molecule comprises detecting a relative or absoluteincrease the level of the molecule. Further aspects of the embodimentsconcern quantifying a galactose-containing molecule in a serum sample.For example, a galactose-containing molecule can be quantified bycomparing an amount of lectin binding to a known reference sample or apanel of reference samples having known concentration agalactose-containing molecule (e.g., samples with known concentrationsof asialohaptoglobin). In certain aspects, such reference samples areassayed simultaneously with the serum sample. However, in some aspects,the reference sample values may be comprised in a reference table.

In further aspects a method of the embodiments further comprisesidentifying a subject as having a colorectal cancer or at risk of havinga colorectal cancer if the subject is determined to have an elevatedlevel of a galactose-containing molecule as compared with a controllevel. In certain aspects, identifying the subject further comprisesreporting that the subject has a colorectal cancer; is at risk of havinga colorectal cancer or has an elevated level of a 40-kDagalactose-containing molecule. For example, the reporting can compriseproviding a written, oral or electronic report. In some aspects, areport is provided to the tested subject, to a health care provider(e.g., a doctor), or to an insurance company.

In yet a further embodiment there is provided a method for monitoringthe effectiveness of an anti-cancer therapy comprising determining thelevel of a galactose-containing molecule (i.e., a molecule that binds toan anti-haptoglobin antibody) in a sample from a subject treated with ananticancer-therapy in accordance with the embodiments. In some aspects,if the levels of the galactose-containing molecule are elevated ascompared to a reference level the subject is in need of additionalanti-cancer therapy. Conversely, if the level the galactose-containingmolecule not elevated as compared to a reference level the subject isnot in need of additional anti-cancer therapy. Thus, in certain aspects,a method is provided for monitoring the effectiveness of an anti-cancertherapy comprising (a) determining the levels of a galactose-containingmolecule in a sample from a subject before and after treatment with ananti-cancer therapy; and (b) identifying the subject as responsive tothe therapy or not responsive to the therapy based on the change inlevels of the galactose-containing molecule. For example, the level of agalactose-containing molecule in a responsive patient should be reducedafter therapy. In further aspects, a patient identified as notresponsive to the first anticancer therapy can be administered a secondanti-cancer therapy.

In some embodiments there is provided a method of treating a subjectcomprising administering an anticancer therapy to a subject identifiedas a having a colorectal cancer by a method of the embodiments. Forexample, the anticancer therapy can be a surgical, radiation,chemotherapeutic, or targeted anticancer therapy.

In still a further embodiment, there is provided a kit comprising atleast a first haptoglobin-binding antibody (e.g., the H-8636 antisera)and a galatose-binding lectin (e.g., Erythrina cristagalli lectin). Incertain aspects, the antibody and/or the lectin is bound to a support orto a detectable label. In a further aspect, a kit of the embodimentscomprises a desialylating reagent such as a neuraminidase enzyme or amild acid, such as a H₂SO₄. Further reagents that can be included in akit of the embodiments include, without limitation, a microtiter plate,a detectable label, a lectin-binding antibody, and a dilution buffer(e.g., PBS or water).

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein. Likewise, aspects of the present embodiments discussed in thecontext of a method quantifying are equally applicable to method fordiagnosing or detecting according to the embodiments.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: A, Sandwich ELISA of asialohaptoglobin prepared by neuraminidasevs. mild acid hydrolysis at 1.25× dilution. B, Sandwich ELISA ofasialohaptoglobin prepared by neuraminidase vs. mild acid hydrolysis at5× dilution.

FIG. 2: Comparison of assay formats. Known serum samples, desialylatedby mild acid hydrolysis at 5× dilution, were assayed with Erythrinacristagalli lectin (ECL) catcher and anti-haptoglobin tracer or withanti-haptoglobin tracer and biotinyl-ECL tracer. Neuraminidase-treatedserum samples were assayed in parallel.

FIG. 3: Constructed receiver operating characteristic curves ofsensitivity versus (1-specificity) using a sandwich ELISA of theembodiments. Results demonstrated that the assay successfullydifferentiated individuals with colorectal neoplasia from normalcontrols with a high degree of sensitivity and specificity. The AUC forthe 40-kDa haptoglobin glycoform (galectin-3 (Gal3) ligand) alone,normal versus cancer, was 0.84 (left panel) and for Gal3 ligand+fecaloccult blood test (FOBT) was 0.91 (right panel).

FIG. 4: Quantitative ELISA detection of the 40-kDa haptoglobin glycoformis highly reproducible. Graphs show assay of serum samples assayed on 3separate plates (with serum order sequential, shuffled, or scrambled) onseparate days (3 sets of 11 plates each in batches of 3 or 4 plates).Net A405 values were used to calculate mg/ml for each assay set(y-axis), which is compared to the median mg/ml for each serum α-axis).

FIG. 5: Graphs show assay of serum samples of separate days (A). Eachplate had triplicate reference standard of 20,000-fold diluted mildacid-treated colon cancer serum (EDRN #73249037, local name C5) andnormal serum (EDRN #73265650, local name N4). B shows the mean of theassay in A.

FIG. 6: Protein concentrations as assessed by a method of theembodiments from samples assayed in 2009 (x-axis) versus 2012 (y-axis).

FIG. 7: Constructed receiver operating characteristic curves ofsensitivity versus (1-specificity) using a bead-based assay of theembodiments.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Embodiments of the invention provide a sensitive and reproducible assayfor the detection of colorectal cancer. In particular, the inventorshave identified a detectable form of a glycoprotein (40-kDa) that bindsto anti-haptoglobin antibodies, but is distinct from other humanhaptoglobin glycoforms, and that is elevated specifically in patientswith colorectal cancer. Because the protein appears to present at somelevel in healthy patients and is immunologically related to haptoglobinthe inventors have developed techniques that allow for quantitativedetection only of specific glycosylated forms of the 40-kDa protein. Theassay involves obtaining a diluted serum sample from patient andtreating the bulk sample to desialylate proteins that are present. Thedesialylated and diluted serum sample is then subjected to a highlysensitive sandwich ELISA assay by capturing the 40-kDa protein using ananti-haptoglobin antibody and detecting the captured protein with adetectable lectin that binds to galactose. By using a desialylated serumsample that has been diluted to between 100-fold and 50,000-fold theassay allows for the amount of the 40-kDa glycoform in a sample to bedetermine quantitatively. An increased level of the 40-kDa glycoform(e.g., as compared to a control sample or a reference level) isindicative of the presence of colorectal cancer in the patient.

The assay systems and methods provided offer significant advantages overconventional methods for detecting colorectal cancer. For example,whereas a colonoscopy is typically only performed one every 5-10 yearsdue to its cost and invasive nature, the blood tests of the embodimentscould be repeated as often as desired by a physician, such as everyyear, twice a year or even every month. Moreover, such small samplevolumes are required for the tests detailed here that portions of bloodtaken for other purposes could be tasked for use in the assay. Theability to repeat these tests frequently and their exquisite sensitivityoffer the opportunity to detect cancers at a very early stage and thusto apply therapy at a time when it can be most effective in improvingpatient outcome.

I. Immunological Reagents

In certain aspects of the invention, one or more antibodies are employedthat bind to the 40-kDa haptoglobin glycoform. Antibodies include anytype of antibody, and specifically refer to antibodies that reactimmunologically with human haptoglobin or immunologically relatedproteins such as the 40-kDa haptoglobin glycoform. In particular, theseantibodies may be used in various diagnostic applications, such as theELISA assay methods detailed below.

As used herein, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE. The term“antibody” is used to refer to any antibody-like molecule that has anantigen binding region, and includes antibody fragments such as Fab′,Fab, F(ab′)2, single domain antibodies (DABs), Fv, scFv (single chainFv), and the like. The techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (see, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; incorporated herein by reference).

Monoclonal antibodies (MAbs) are recognized to have certain advantages,e.g., reproducibility and large-scale production. The invention thusprovides monoclonal antibodies of the human, murine, monkey, rat,hamster, rabbit and even chicken origin. Due to the ease of preparationand ready availability of reagents, murine monoclonal antibodies willoften be utilized.

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal with,for example a human haptoglobin (e.g., a purified haptoglobin)composition in accordance with the present invention and collectingantisera from that immunized animal.

A wide range of animal species can be used for the production ofantisera. Typically the animal used for production of antisera is arabbit, a mouse, a rat, a hamster, a guinea pig or a goat. The choice ofanimal may be decided upon the ease of manipulation, costs or thedesired amount of sera, as would be known to one of skill in the art.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitableadjuvants include all acceptable immunostimulatory compounds, such ascytokines, chemokines, cofactors, toxins, plasmodia or syntheticcompositions.

Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12,interferon, GM-CSF, BCG, aluminum hydroxide, MDP compounds, such asthur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A(MPL). RIBI, which contains three components extracted from bacteria,MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2%squalene/Tween 80 emulsion is also contemplated. MHC antigens may evenbe used. Exemplary adjuvants include complete Freund's adjuvant (anon-specific stimulator of the immune response containing killedMycobacterium tuberculosis), incomplete Freund's adjuvants and aluminumhydroxide adjuvant.

In addition to adjuvants, it may be desirable to coadminister biologicresponse modifiers (BRM), which have been shown to upregulate T cellimmunity or down-regulate suppressor cell activity. Such BRMs include,but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, Pa.);low-dose Cyclophosphamide (CYP; 300 mg/m²) (Johnson/Mead, NJ), cytokinessuch as -interferon, IL-2, or IL-12 or genes encoding proteins involvedin immune helper functions, such as B-7.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen including but not limited to subcutaneous, intramuscular,intradermal, intraepidermal, intravenous and intraperitoneal. Theproduction of polyclonal antibodies may be monitored by sampling bloodof the immunized animal at various points following immunization.

A second, booster dose (e.g., provided in an injection), may also begiven. The process of boosting and titering is repeated until a suitabletiter is achieved. When a desired level of immunogenicity is obtained,the immunized animal can be bled and the serum isolated and stored,and/or the animal can be used to generate MAbs.

For production of rabbit polyclonal antibodies, the animal can be bledthrough an ear vein or alternatively by cardiac puncture. The removedblood is allowed to coagulate and then centrifuged to separate serumcomponents from whole cells and blood clots. The serum may be used as isfor various applications or else the desired antibody fraction may bepurified by well-known methods, such as affinity chromatography usinganother antibody, a peptide bound to a solid matrix, or by using, e.g.,protein A or protein G chromatography. Thus, in certain aspects,polyclonal antibodies for used according to the embodiments are purifiedantibodies, such an IgG fraction of antibodies.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified protein, polypeptide, peptide or domain, be it awild-type or mutant composition. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells. In some embodiments, however, the antibody that reactsimmunologically with the anti-tumor antigen antibody and/or theanti-tumor antigen antibody are present endogenously in a subject.

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Rodents such as mice and rats are often used, however, the use ofrabbit, sheep or frog cells is also possible.

The animals are injected with antigen, generally as described above. Theantigen may be mixed with adjuvant, such as Freund's complete orincomplete adjuvant. Booster administrations with the same antigen orDNA encoding the antigen would occur at approximately two-weekintervals.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible.

Often, a panel of animals will have been immunized and the spleen of ananimal with the highest antibody titer will be removed and the spleenlymphocytes obtained by homogenizing the spleen with a syringe.Typically, a spleen from an immunized mouse contains approximately 5×10⁷to 2×10⁸ lymphocytes.

The antibody producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized Myeloma cell lines suited foruse in hybridoma producing fusion procedures preferably are non antibodyproducing, have high fusion efficiency, and enzyme deficiencies thatrender then incapable of growing in certain selective media whichsupport the growth of only the desired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83,1984). For example, where the immunized animal is a mouse, one may useP3 X63/Ag8, X63 Ag8.653, NS1/1.Ag 4 1, Sp210 Ag14, FO, NSO/U, MPC 11,MPC11×45 GTG 1.7 and S194/5XXO Bul; for rats, one may use R210.RCY3, Y3Ag 1.2.3, IR983F and 4B210; and U 266, GM1500 GRG2, LICR LON HMy2 andUC729 6 are all useful in connection with human cell fusions.

One particular murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the 8azaguanine resistant mouse murine myeloma SP2/0 non producer cell line.

Methods for generating hybrids of antibody producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al., (1977). The use ofelectrically induced fusion methods is also appropriate (Goding pp.71-74, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryagents are aminopterin, methotrexate, and azaserine. Aminopterin andmethotrexate block de novo synthesis of both purines and pyrimidines,whereas azaserine blocks only purine synthesis. Where aminopterin ormethotrexate is used, the media is supplemented with hypoxanthine andthymidine as a source of nucleotides (HAT medium). Where azaserine isused, the media is supplemented with hypoxanthine.

The favored selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. First, a sample of the hybridomacan be injected (often into the peritoneal cavity) into ahistocompatible animal of the type that was used to provide the somaticand myeloma cells for the original fusion (e.g., a syngeneic mouse).Optionally, the animals are primed with a hydrocarbon, especially oilssuch as pristane (tetramethylpentadecane) prior to injection. Theinjected animal develops tumors secreting the specific monoclonalantibody produced by the fused cell hybrid. The body fluids of theanimal, such as serum or ascites fluid, can then be tapped to provideMAbs in high concentration. Second, the individual cell lines could becultured in vitro, where the MAbs are naturally secreted into theculture medium from which they can be readily obtained in highconcentrations.

MAbs produced by either means may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asHPLC or affinity chromatography. Fragments of the monoclonal antibodiesof the invention can be obtained from the monoclonal antibodies soproduced by methods which include digestion with enzymes, such as pepsinor papain, and/or by cleavage of disulfide bonds by chemical reduction.Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer.

It is also contemplated that a molecular cloning approach may be used togenerate monoclonals. In one embodiment, combinatorial immunoglobulinphagemid libraries are prepared from RNA isolated from the spleen of theimmunized animal, and phagemids expressing appropriate antibodies areselected by panning using cells expressing the antigen and controlcells. The advantages of this approach over conventional hybridomatechniques are that approximately 10⁴ times as many antibodies can beproduced and screened in a single round, and that new specificities aregenerated by H and L chain combination which further increases thechance of finding appropriate antibodies.

Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer, orby expression of full-length gene or of gene fragments in E. coli.

II. Lectins

It has long been known that extracts from certain plants couldagglutinate red blood cells. Although the term “lectin” was originally aterm used to describe agglutinins which could discriminate among typesof red blood cells. However, the term is used more defined assugar-binding proteins from a wide variety of sources. Lectins have beenfound in plants, viruses, microorganisms and animals. Although lectinsshare the common property of binding to defined sugar structures, theirroles in various organisms are not likely to be the same and remainincompletely understood.

Because of the specificity that each lectin has toward a particularcarbohydrate structure, even oligosaccharides with identical sugarcompositions can be distinguished or separated. Some lectins will bindonly to structures with mannose or glucose residues, while others mayrecognize only galactose residues. Some lectins require that theparticular sugar be in a terminal non-reducing position in theoligosaccharide, while others can bind to sugars within theoligosaccharide chain. Some lectins do not discriminate between alphaand beta anomers, while others require not only the correct anomericstructure but a specific sequence of sugars for binding. The affinitybetween a lectin and its receptor may vary a great deal due to smallchanges in the carbohydrate structure of the receptor.

Thus, lectins can be used in similar detection methods as antibodies,for the detection of specific carbohydrate moieties. Embodiments of thepresent invention provide assays for the detection of a 40-kDahaptoglobin glycoform using lectins as selective binding agents.Generally, the carbohydrate composition of the 40-kDa glycoform isexploited in order to detect its presence in a sample. For example,embodiments of the methods of the invention employ galactose-bindinglectins to capture or detect the 40-kDa protein comprising such agalactose moiety.

In the study detailed here, the inventors used galactose binding lectinsto detect the 40-kDa glycoform in desialylating the diluted serum.Lectins that were found to be effective include mammalian galectin-3,Ricinus communis lectin, Datura stramonium lectin, Erythrina cristagallilectin, and Lycopersicon esculentum lectin. Of these however, thehighest specificity was achieved with Erythrina cristagalli lectin.

In some aspects, lectins for use according to the embodiments arelabeled. Methods for labeling antibodies can generally also be appliedto lectins and are further detailed below.

III. Antibody and Lectin Conjugates

The present invention further provides antibodies and lectins reactivewith human haptoglobin and the 40-kDa haptoglobin glycoform, that arelinked to at least one agent to form an antibody or lectin conjugate. Inorder to increase the efficacy of such conjugates as diagnostic agents,it is conventional to link or covalently bind or complex at least onedesired molecule or moiety. Such a molecule or moiety may be, but is notlimited to, at least one reporter molecule. Any antibody or lectin ofsufficient selectivity, specificity or affinity may be employed as thebasis for a conjugate. Such properties may be evaluated usingconventional immunological screening methodology known to those of skillin the art. It will therefore be understood that embodiments referringto antibody conjugates are thus equally applicable to lectin conjugatesand vice versa.

A. Conjugation

Thus, in certain aspects a lectin antibody of the embodiments isconjugated to a reporter. Any of a wide array of conjugation schemes canbe employed to linkage of an antibody of lectin to a reporter as furtherdetailed below.

An exemplary hetero-bifunctional cross-linker contains two reactivegroups: one reacting with primary amine group (e.g., N hydroxysuccinimide) and the other reacting with a thiol group (e.g., pyridyldisulfide, maleimides, halogens, etc.). Through the primary aminereactive group, the cross-linker may react with the lysine residue(s) ofone protein (e.g., the selected antibody or lectin) and through thethiol reactive group, the cross-linker, already tied up to the firstprotein, reacts with the cysteine residue (free sulfhydryl group) of theother protein (e.g., the reporter). In some cases, it is preferred thata cross-linker having reasonable stability in serum samples will beemployed. Numerous types of disulfide-bond containing linkers are knownthat can be successfully employed to conjugate targeting andtherapeutic/preventative agents. These linkers are thus one group oflinking agents.

Another cross-linking reagent is SMPT, which is a bifunctionalcross-linker containing a disulfide bond that is “sterically hindered”by an adjacent benzene ring and methyl groups. It is believed thatsteric hindrance of the disulfide bond serves a function of protectingthe bond from attack by thiolate anions such as glutathione which can bepresent in tissues and blood, and thereby help in preventing decouplingof the conjugate prior to the delivery of the attached agent to thetarget site.

The SMPT cross-linking reagent, as with many other known cross-linkingreagents, lends the ability to cross-link functional groups such as theSH of cysteine or primary amines (e.g., the epsilon amino group oflysine). Another possible type of cross-linker includes thehetero-bifunctional photoreactive phenylazides containing a cleavabledisulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido)ethyl-1,3′-dithiopropionate. The N-hydroxy-succinimidyl group reactswith primary amino groups and the phenylazide (upon photolysis) reactsnon-selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers also can beemployed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1988). The use of suchcross-linkers is well understood in the art. Another embodiment involvesthe use of flexible linkers.

U.S. Pat. No. 4,680,338, describes bifunctional linkers useful forproducing conjugates of ligands with amine-containing polymers and/orproteins, especially for forming antibody conjugates with chelators,drugs, enzymes, detectable labels and the like. U.S. Pat. Nos. 5,141,648and 5,563,250 disclose cleavable conjugates containing a labile bondthat is cleavable under a variety of mild conditions. This linker isparticularly useful in that the agent of interest may be bonded directlyto the linker, with cleavage resulting in release of the active agent.Preferred uses include adding a free amino or free sulfhydryl group to aprotein, such as an antibody, or a drug.

U.S. Pat. No. 5,856,456 provides peptide linkers for use in connectingpolypeptide constituents to make fusion proteins, e.g., single chainantibodies. The linker is up to about 50 amino acids in length, containsat least one occurrence of a charged amino acid (preferably arginine orlysine) followed by a proline, and is characterized by greater stabilityand reduced aggregation. U.S. Pat. No. 5,880,270 disclosesaminooxy-containing linkers useful in a variety of immunodiagnostic andseparative techniques.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter & Haley, 1983). Inparticular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and may be used asantibody binding agents.

Some attachment methods involve the use of a metal chelate complexemploying, for example, an organic chelating agent such adiethylenetriaminepentaacetic acid anhydride (DTPA);ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/ortetrachloro-3-6-diphenylglycouril-3 attached to the antibody (U.S. Pat.Nos. 4,472,509 and 4,938,948, each incorporated herein by reference).Antibodies or lectins may also be reacted with an enzyme in the presenceof a coupling agent such as glutaraldehyde or periodate. Conjugates withfluorescein markers are prepared in the presence of these couplingagents or by reaction with an isothiocyanate. In U.S. Pat. No.4,938,948, imaging of breast tumors is achieved using monoclonalantibodies and the detectable imaging moieties are bound to the antibodyusing linkers such as methyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

B. Reporter Molecules

A reporter molecule is defined as any moiety which may be detected usingan assay. Non-limiting examples of reporter molecules which have beenconjugated to antibodies or lectins include enzymes, radiolabels,haptens, fluorescent labels, phosphorescent molecules, chemiluminescentmolecules, chromophores, luminescent molecules, photoaffinity molecules,colored particles or ligands, such as biotin.

In the case of radioactive isotopes that can be conjugated to antibodies(or lectins) for diagnostic applications, one might mention astatine²¹¹,¹⁴carbon, ⁵¹chromium, ³⁶-chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu,gallium⁶⁷, ³hydrogen, iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹,⁵⁹iron, ³²phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur,technicium^(99m) and/or yttrium⁹⁰. Radioactively labeled antibodies ofthe present invention may be produced according to well-known methods inthe art. For instance, antibodies or lectins can be iodinated by contactwith sodium and/or potassium iodide and a chemical oxidizing agent suchas sodium hypochlorite, or an enzymatic oxidizing agent, such aslactoperoxidase. Antibodies or lectins according to the invention may belabeled with technetium^(99m) by ligand exchange process, for example,by reducing pertechnate with stannous solution, chelating the reducedtechnetium onto a Sephadex column and applying the antibody to thiscolumn.

Alternatively, direct labeling techniques may be used, e.g., byincubating pertechnate, a reducing agent such as SNCl₂, a buffersolution such as sodium-potassium phthalate solution, and the antibody.Intermediary functional groups which are often used to bindradioisotopes which exist as metallic ions to antibody arediethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraceticacid (EDTA).

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed.

Another type of conjugate contemplated in the present invention arethose where the antibody or lectin is linked to a secondary bindingligand and/or to an enzyme (an enzyme tag) that will generate a coloredproduct upon contact with a chromogenic substrate. Examples of suitableenzymes include urease, alkaline phosphatase, (horseradish) hydrogenperoxidase or glucose oxidase. Secondary binding ligands are biotinand/or avidin and streptavidin compounds. The use of such labels is wellknown to those of skill in the art and are described, for example, inU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149 and 4,366,241; each incorporated herein by reference.

Likewise, in certain applications it is desirable to immobilize andantibody on a solid surface. For example, antibodies can be absorbed tothe surface of the wells of a plate. Such absorption can rely onnon-specific interactions with the polymers of the plate or can involvespecific binding of the antibodies (e.g., by protein A). In certainaspects antibodies are covalently linked to surface, for example byUV-cross linking.

Optical imaging with dyes also permit visualization of biologicalactivities (Blasdel et al., 1986; Grinvald et al., 1988; Kauer et al.,1988; Lieke et al., 1989). Dyes that are sensitive to physicochemicalenvironments (such as pressure, cell membrane potential, ionconcentration, acidity, partial pressure of oxygen, etc.), are subjectto changes in absorption or emission of light. The resulting changes actas optical probes to transform biological activities into opticalsignals that can be converted into optical images.

Water soluble dyes are particularly well suited, including acid dyes,basic dyes, direct dyes, and so on, and equivalents thereof. The dyecomposition may be prepared as a dry material for ease of storage andpackaging. If prepared as a dry composition, prior to usage thecomposition may be prepared as a solution using a suitable liquid,including water and various organic solvents, or mixtures thereof and soon, by techniques well known to those skilled in the art.

Dyes include methylene blue, Tartrazine (CI 19140), Quinoline Yellow (CI47005), Eosin (CI 45380), Acid Phloxine (CI 45410), Erythrosine (CI45430), Sunset Yellow FCF (CI 15985), Acid Violet 5B (CI 42640), PatentBlue AF (CI 42080), Brilliant Cyanine 6B (CI 42660), Acid Brilliant BlueFCF (CI 42090), Naphthalene Green VSC(CI 44025) and Acid Blue Black 10B(CI 20470); and direct dyes such as Paper Yellow GG (CI Direct Yellow131), Direct Scarlet 4BS (CI 29160), Congo Red (CI 22120), Violet BB (CI27905), Direct Sky Blue 5B (CI 24400), Patent Blue Violet, Sulfan Dye),Pentamine, guajazulen blue Pentamine, Phthalocyanine Blue (CI 74180),Black G (CI 35255) and Deep Black XA (CI Direct Black 154). The CInumber in the description above indicates the identification number inthe Color Index, 3rd Ed., The Society of Dyers and Colorists, Bradford,Yorkshire (1971). Preferred dyes include Isosulfan blue or other dyewhich travels through the lymphatic system.

Chromophores include Fluorescein, Rhodamine, Acid Fuchsin; AcridineOrange; Acridine Red; Acridine Yellow; Alizarin Red; Allophycocyanin;Astrazon Brilliant; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow;Bodipy Fl; Bodipy TMR; Bodipy TR; Calcein; Calcein Blue; Calcium Green;Calcium Orange; Calcofluor White; Cascade Blue; Flazo Orange;Fluorescein Isothiocyanate (FITC); Fura-2; Fura Red; Genacryl BrilliantRed B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl YellowSGF; Granular Blue; Lucifer Yellow CH; Lucifer Yellow VS; LysoSensorBlue DND-192, DND-167; LysoSensor Green DND-153, DND-189; LysoTrackerGreen; LysoTracker Yellow; LysoTracker Red; Magdala Red; MagnesiumGreen; Magnesium Orange; Mitotracker Green FM; Mitotracker Orange; NileRed; Nuclear Fast Red; Nuclear Yellow; Oregon Green 488; Oregon Green500; Oregon Green 514; Phorwite AR; Phorwite BKL; Phorwite Rev; PhorwiteRPA; Pontochrome; Blue Black; Procion Yellow; Pyrozal Brilliant;Rhodamine Green; Rhodamine Red; Rhodol Green Fluorophore; Rose Bengal;Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant RedB; Sevron Orange; Sevron Yellow L; Texas Red; Thiozol Orange; True Blue;and Xylene Orange.

IV. Immunodetection Methods

In certain embodiments, the present invention concerns immunodetectionmethods for binding, purifying, removing, quantifying and/or otherwisegenerally detecting biological components such as a 40-kDa haptoglobinglycoform. Some immunodetection methods include enzyme linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometricassay, fluoroimmunoassay, chemiluminescent assay and bioluminescentassay to mention a few. The steps of various useful immunodetectionmethods have been described in the scientific literature, such as, e.g.,Doolittle and Ben-Zeev, 1999; Gulbis and Galand, 1993; De Jager et al.,1993; and Nakamura et al., 1987, each incorporated herein by reference.

In general, the immunobinding methods include obtaining a samplesuspected of containing a 40-kDa haptoglobin glycoform, and contactingthe sample with a first antibodies that react immunologically with the40-kDa glycoform in accordance with the present invention, as the casemay be, under conditions effective to allow the formation ofimmunocomplexes.

These methods include methods for purifying a 40-kDa haptoglobinglycoform tissue or organism's samples. In these instances, the antibodyremoves the 40-kDa protein (and haptoglobin) that react immunologicallywith antibody. The antibody will preferably be linked to a solidsupport, such as in the form of a column matrix or the well of a plate,and the sample suspected of containing the 40-kDa haptoglobin glycoformwill be applied to the immobilized antibody. The unwanted componentswill be washed from the column, leaving the antigen immunocomplexed tothe immobilized antibody.

The immunobinding methods also include methods for detecting andquantifying the amount of the 40 kDa haptoglobin glycoform in a sampleand the detection and quantification of any immune complexes formedduring the binding process. Here, one would obtain a sample suspected ofcontaining an antigen, and contact the sample with an antibody againstthe antigen, and then detect and quantify the amount of immune complexesformed under the specific conditions.

In terms of antigen detection, the biological sample analyzed may be anysample that is suspected of containing an antigen, such as, for example,a tissue section, or specimen, a homogenized tissue extract, a cell, anorganelle, separated and/or purified forms of any of the aboveantigen-containing compositions, or even any biological fluid that comesinto contact with the cell or tissue, including blood and/or serum,although tissue samples or extracts may be used.

Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, anyantibodies that react immunologically with anti-tumor antigen antibodiespresent. After this time, the sample-antibody composition, such as atissue section, ELISA plate or dot blot, will generally be washed toremove any non-specifically bound protein species.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. Patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

The antibody or lectin employed in the detection may itself be linked toa detectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary immune complexes in thecomposition to be determined. Alternatively, the first antibody orlectin that becomes bound within the primary immune complexes may bedetected by means of a second binding ligand that has binding affinityfor the antibody. In these cases, the second binding ligand may belinked to a detectable label. The second binding ligand is itself oftenan antibody, which may thus be termed a “secondary” antibody. Theprimary immune complexes are contacted with the labeled, secondarybinding ligand, or antibody, under effective conditions and for a periodof time sufficient to allow the formation of secondary immune complexes.The secondary immune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the antibody or lectin is used to formsecondary immune complexes, as described above. After washing, thesecondary immune complexes are contacted with a third binding ligand orantibody that has binding affinity for the second antibody, again undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (tertiary immune complexes). The thirdligand or antibody is linked to a detectable label, allowing detectionof the tertiary immune complexes thus formed. This system may providefor signal amplification if this is desired.

One method of immunodetection designed by Charles Cantor uses twodifferent antibodies. A first step biotinylated, monoclonal orpolyclonal antibody is used to detect the target antigen(s), and asecond step antibody is then used to detect the biotin attached to thecomplexed biotin. In that method the sample to be tested is firstincubated in a solution containing the first step antibody. If thetarget antigen is present, some of the antibody binds to the antigen toform a biotinylated antibody/antigen complex. The antibody/antigencomplex is then amplified by incubation in successive solutions ofstreptavidin (or avidin), biotinylated DNA, and/or complementarybiotinylated DNA, with each step adding additional biotin sites to theantibody/antigen complex. The amplification steps are repeated until asuitable level of amplification is achieved, at which point the sampleis incubated in a solution containing the second step antibody againstbiotin. This second step antibody is labeled, as for example with anenzyme that can be used to detect the presence of the antibody/antigencomplex by histoenzymology using a chromogen substrate. With suitableamplification, a conjugate can be produced which is macroscopicallyvisible.

Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. At leastin theory, the enormous amplification capability and specificity of PCRcan be utilized to detect a single antigen molecule.

The immunodetection methods of the present invention have evidentutility in the diagnosis and prognosis of conditions such as cancerwherein a specific tumor antigen is expressed, and wherein antibodiesexist that react immunologically to an anti-tumor antigen antibody Here,a biological and/or clinical sample suspected of containing a specificdisease associated antibody is used. However, these embodiments alsohave applications to non-clinical samples, such as in the titering ofantigen or antibody samples, for example in the selection of hybridomas.

In the clinical diagnosis and/or monitoring of patients with variousforms a disease, such as, for example, colorectal cancer, the detectionof a cancer specific antigens that react and/or an alteration in thelevels of such an antigen, in comparison to the levels in acorresponding biological sample from a normal subject is indicative of apatient with cancer. However, as is known to those of skill in the art,such a clinical diagnosis would not necessarily be made on the basis ofthis method in isolation. Those of skill in the art are very familiarwith differentiating between significant differences in types and/oramounts of biomarkers, which represent a positive identification, and/orlow level and/or background changes of biomarkers. Indeed, backgroundexpression levels are often used to form a “cut-off” above whichincreased detection will be scored as significant and/or positive. Ofcourse, the antibodies of the present invention in any immunodetectionor therapy known to one of ordinary skill in the art.

ELISAs

As detailed above, immunoassays, in their most simple and/or directsense, are binding assays. Certain immunoassays are the various types ofenzyme linked immunosorbent assays (ELISAs) and/or radioimmunoassays(RIA).

In some aspects of the invention, there are ELISA assays, including inkits, to test samples of subjects that are suspect or at risk for thedevelopment of colorectal cancer. In one exemplary ELISA, theanti-haptoglobin antibodies of the invention are immobilized onto aselected surface exhibiting protein affinity, such as a well in apolystyrene microtiter plate. Then, a test composition suspected ofcontaining the antigen, such as a diluted clinical sample, is added tothe wells. After binding and/or washing to remove non-specifically boundimmune complexes, the bound antigen may be detected. Detection can beachieved by contacting the sample with a galactose-binding lectin thatis linked to a detectable label. This type of ELISA is a “sandwichELISA”. Detection may also be achieved by the addition of agalatose-binding lectin, followed by the addition of a further antibodythat has binding affinity for the lectin, with the further antibodybeing linked to a detectable label.

In a further exemplary ELISA, the galactose-binding lectins of theinvention are immobilized onto a selected surface exhibiting proteinaffinity, such as a well in a polystyrene microtiter plate. Then, a testcomposition suspected of containing the antigen, such as a dilutedclinical sample, is added to the wells. After binding and/or washing toremove non-specifically bound immune complexes, the bound antigen may bedetected. Detection can be achieved by contacting the sample with ahaptoglobin-binding antibody that is linked to a detectable label.Detection may also be achieved by the addition of a haptoglobin-bindingantibody, followed by the addition of a further antibody that hasbinding affinity for the haptoglobin-binding antibody (e.g., a secondaryantibody), with the further antibody being linked to a detectable label.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either lectin or antibody (e.g., ananti-haptoglobin antibody), one will generally incubate the wells of theplate with a solution of the lectin or antibody, either overnight or fora specified period of hours, such as for 4, 5, 6, 7, 8, 9 or 10 hours.Likewise, the incubation can be performed at a specified temperature,such as between about 1° C. and 22° C., e.g., at 4° C. The wells of theplate will then be washed to remove incompletely adsorbed material. Anyremaining available surfaces of the wells are then “coated” with anonspecific protein that is antigenically neutral with regard to thetest antisera. These include bovine serum albumin (BSA), casein orsolutions of milk powder. The coating allows for blocking of nonspecificadsorption sites on the immobilizing surface and thus reduces thebackground caused by nonspecific binding of antisera onto the surface.

After binding of a lectin or antibody to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with thebiological sample to be tested under conditions effective to allowimmune complex (antigen/antibody) formation. For example, the reactioncould be incubated for 1, 2, 3, 4, or more hours at room temperature.Detection of the immune complex then requires a labeled secondarybinding ligand (e.g., a lectin) or antibody, or a secondary bindingligand or antibody in conjunction with a labeled tertiary antibody or athird binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and/or antibodies with solutions such as BSA, bovine gammaglobulin (BGG) or phosphate buffered saline (PBS)/Tween. These addedagents also tend to assist in the reduction of nonspecific background.For example, the inclusion of a detergent such as 0.01 to 0.1% TWEEN-20can significantly reduce non-specific background.

The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 20° C. to 27° C., or insome cases overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. A particular washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the lectin, second or third antibody willhave an associated label to allow detection. In some cases this will bean enzyme that will generate color development upon incubating with anappropriate chromogenic substrate. In certain aspects the label is anaffinity label such a biotin and detection can be achieved by furthercontact with a detectable affinity molecules (e.g., a labeled avadin).Thus, for example, one will desire to contact or incubate the first andsecond complex with a urease, glucose oxidase, alkaline phosphatase orhydrogen peroxidase-conjugated antibody for a period of time and underconditions that favor the development of further immune complexformation (e.g., incubation for 2 hours at room temperature in aPBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea, or bromocresolpurple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS),or H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generated, e.g., usinga visible spectra spectrophotometer.

EXAMPLES

The following examples are included to demonstrate particularembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constituteparticular modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

Example 1 40-kDa Haptoglobin Glycoform Detection by ELISA Assay Format

Initial results were obtained using a Western blot format to quantify a40-kDa haptoglobin glycoform and galectin-3 ligand in sera, however thisformat was not suitable for a diagnostic assay. For example,disadvantages of initial assay include that it was very time-consumingand did not yield a linear response that could be used to quantify the40 kDa haptoglobin glycoform in a sample, i.e., 40-kDa band density wasnot directly proportional to amount of the glycoform. Likewise theWestern blot assay exhibited gel-to-gel variability and required a knownpositive cancer serum as a common reference serum, which is notpractical for scale-up.

A sandwich assay was developed to allow measurement of the 40-kDaglycoform in a complex background by immobilizing ligand with a“catcher” antibody or lectin and detecting the ligand with a “tracer”antibody or ligand. Two complementary sandwich ELISA formats usinganti-haptoglobin and lectin were extensively evaluated. A sandwichenzyme-linked immunosorbent assay using lectin catcher andanti-haptoglobin tracer was employed in early developmental studies.However, the alternative sandwich ELISA, using anti-haptoglobin catcherand lectin tracer was found to be superior in discriminating knowncancer sera from normal specimens, and was therefore selected forfurther development and clinical validation (FIG. 2).

Lectin Selection

Initial attempts were made to develop a lectin/anti-haptoglobin sandwichELISA assay using biotinyl-galectin-3. Although biotinyl-galectin-3 waseffective for detection of the 40-kDa glycoform on Western blots, it wasnot sufficiently sensitive to use as a reagent for the sandwich ELISA.It was found that the failure of biotinyl-galectin-3 to bind toasialohaptoglobin in a microplate format reflected a loss ofcarbohydrate binding, and was not due to gross degradation of galectin-3or to inability of secondary reagents to detect biotinyl-galectin-3.

Preliminary studies indicated that Erythrina cristagalli lectin (ECL)had a specificity similar to the mammalian lectin galectin-3. Therefore,ECL was used to develop a sandwich ELISA. Both the ECL/anti-haptoglobinand anti-haptoglobin/biotinyl-ECL formats detected asialo-haptoglobin inserum samples, but the anti-haptoglobin/biotinyl-ECL sandwich ELISA wasmore specific for known cancer specimens. Otherbeta-galactoside-specific plant lectins that were tested included thosefrom Ricinus communis, Sophora japonica, Datura stramonium, andLycopersicon esculentum. However, none of these appeared superior to ECL(Table 1).

TABLE 1 Sensitivity and specificity of beta-galactoside specific lectinsin sandwich ELISAs. Lectin Sensitivity Specificity Accuracy Erythrinacristagalli 70% 50% 60% Ricinus communis 40% 40% 40% Datura stramonium60% 60% 60% Lycopersicon esculentum 40% 60% 50%

One key step necessary for the successful development of the sandwichELISA is the method used for desialylation. Various studies wereundertaken to address the necessity for desialylation, the relativeefficacy of neuraminidase enzyme vs. mild acid, and the effect ofdilution prior to mild acid treatment.

Desialylation

Initial studies established that desialylation was necessary for bindingof galectin-3 to the 40 kDa glycoform, but a desialylation procedureneed to be developed that could yield quatitaive assay results. Indeveloping the sandwich ELISA, it was found that desialylation wasnecessary for binding of ECL to haptoglobin. Initially, desialylationwas performed either with Vibrio cholerae neuraminidase digestion orwith mild acid hydrolysis (0.1 NH₂SO₄, 60 min at 80° C.) with minimaldilution (1.25-fold). Sandwich ELISAs, using either ECL/anti-haptoglobinor anti-haptoglobin/ECL indicated that the neuraminidase digestion gaveat least a 10-fold higher signal than the mild acid hydrolysis treatment(FIG. 1). Therefore, early assays of known cancer and normal sera wereperformed using neuraminidase digestion, but there was difficulty ingetting reproducible results.

To further develop the desialylation method, the loss of antigenicity ofhaptoglobin caused by mild acid treatment was studied in detail. Directbinding assays established that this was due to loss of binding ofanti-haptoglobin to the mild-acid-treated glycoprotein. Some of thisloss of antigenicity could be due to concentration-dependentaggregation, thus, the mild acid treatment was repeated at higherdilutions (5-fold, 25-fold, and finally 100-fold dilution). With themild acid hydrolysis performed at higher dilutions, signal wasequivalent to that obtained with neuraminidase digestion, with a higherspecificity for cancer sera (FIG. 2). Desialylation by mild acidhydrolysis at a 100-fold dilution was therefore used in clinical methodvalidation.

Additional Element of the ELISA

Two different anti-haptoglobin antibodies were evaluated for potentialuse in sandwich ELISA, a polyclonal anti-haptoglobin and a mousemonoclonal anti-haptoglobin. The two antibodies gave similar results inthe ECL/anti-haptoglobin sandwich ELISA format, but only the polyclonalanti-haptoglobin could practically be used in theanti-haptoglobin/biotinyl-ECL format.

Concentrations of anti-haptoglobin used as catcher, or biotinyl-ECL usedas tracer, and of ABC complex were varied to ensure they were notlimiting. In addition, the effects of blocking with a commercial blocksolution rather than with bovine serum albumin (BSA) and of dilutingsera in PBS rather than PBST, was studied, however, BSA and PSBT werefound to be superior. Extensive studies with known cancer sera and knownnormal sera were also performed to determine the optimal 20,000-folddilution of desialylated serum that would fall within the linear rangeof the assay.

Example 2 Detailed 40-kDa Haptoglobin Glycoform Detection Protocol MildAcid Hydrolysis and Dilution:

For initial sample preparation the following steps were used:

-   -   Serum or plasma specimens were thawed (aliquotted in small        volumes (0.3 ml), then stored at −80° C.).    -   5 μl samples of coded serum were collected. Residual aliquots        were marked as once-thawed, and return excess to −80° C.    -   Samples were added to 395 μl water in 1.5 ml tube and mixed.    -   100 μl 0.5 NH₂SO₄ was added and mixed with the diluted samples.    -   Samples were heated for 60 min at 80° C. and then cooled on ice.    -   100 μl 10×PBS was added and mixed with the samples.    -   100 μl 0.5 N NaOH was added and mixed with the samples.    -   300 μl of water was added and mixed with the samples    -   The pH of the samples were checked (with pH paper) and recorded.    -   Resulting 200-fold diluted desialylated serum samples were        refrozen in 200 μl aliquots at −20° C.    -   Three aliquots are used on separate days for ELISA. Two aliquots        are reserved.

Preparation of Anti-Haptoglobin Coated Plates: Day 1

-   -   Four 96-well microtiter plates were marked with triplicate wells        inside for serum specimens, PBST blanks and normal        asialohaptoglobin standards. Typical plate geometry is shown in        Table 2.    -   100 μl Rabbit anti-haptoglobin (Sigma Catalog #H-8636) was        diluted into 25 ml PBS and 50 μl was added to each well of four        microtiter plates. The plate is left overnight at 4° C.

Day 2

-   -   Anti-haptoglobin is decanted and discarded. Excess liquid is        removed by striking inverted plate on paper towels. Each well is        then washed once with PBS using a squeeze bottle. The wash is        decanted and discarded. Again excess liquid is removed by        striking inverted plate on paper towels.    -   200 μl of 1% BSA in PBS (prepared fresh daily) was added to each        well and the components left for 60 min at room temp.        (Meanwhile, final dilutions of standards, unknowns, and controls        were prepared)    -   BSA/PBS was decanted and discarded. Excess liquid is removed by        striking inverted plate on paper towels.

Final Dilutions of Standards, Unknowns, and Controls.

Final dilutions of coded desialylated serum were produced by thawingfresh aliquots of 56 coded specimens of 200-fold diluted desialylatedserum. 5 μl samples were taken and the residual aliquot was marked asonce-thawed, and return to −20° C. The 5 μl sample added and mixed withto 495 μl of PBST on ice in a 500 μl tube to give 20,000-fold finaldilution relative to serum.

Final dilution of known positive and negative specimens were produced bythawing re-frozen aliquots of 2 known positive cancer specimens(200-fold diluted desialylated serum) or re-frozen aliquots of 2 knownnegative normal specimens (200-fold diluted desialylated serum),respectively. 10 μl samples were removed from each specimen and theresidual aliquots returned to −20° C. The 10 μl samples were each addedto and mixed with 990 μl PBST on ice in 1.5 ml tubes to give 20,000-foldfinal dilution relative to serum.

Final dilution of normal asialohaptoglobin standards were produced bythawing 50 μg/ml stock of normal asialohaptoglobin (asHP). Aftersampling, the residual stock was returned to −20° C. A 20 μl of 50 μg/mlasHP was added and mixed with 1980 μl PBST in 15 ml tube to give 500ng/ml asHP. A 400 μl sample of 500 ng/ml asHP was added and mixed with600 μl PBST in 1.5 ml tube to give 200 ng/ml asHP. A 200 μl sample of500 ng/ml asHP was added to and mixed with 800 μl PBST in 1.5 ml tube togive 100 ng·ml asHP. A 100 μl sample of 500 ng/ml asHP was added to andmixed with 900 μl PBST in 1.5 ml tube to give 50 ng/ml asHP. A 40 μlsample of 500 ng/ml asHP was added to and mixed with 960 μl PBST in 1.5ml tube to give 20 ng/ml asHP. Finally, a 20 μl sample of 500 ng/ml asHPwas added to and mixed with 980 μl PBST in 1.5 ml tube to give 10 ng·mlasHP.

Binding of Analytes and Standards:

-   -   50 μl/well of 20,000-fold diluted unknown desialylated serum in        PBST was added in triplicate (3×16=48 interior wells/plate).    -   50 μl/well of 20,000-fold diluted known control sera in PBST was        added in triplicate (3×4=12 wells/plate).    -   50 μl/well of PBST was added to 24 wells (6 interior & 18        exterior) as blanks.    -   50 μl/well of asHP standards (10 ng/ml, 20 ng/ml, 50 ng/ml, 100        ng/ml, 200 ng/ml, & 500 ng/ml) was added, in triplicate (3×6=18        exterior wells/plate).

Typical plate geometry is shown in Table 2.

-   -   The plate is incubated for 1 h at room temp. (Meanwhile, 1 ug/ml        biotinyl Erythrina cristagalli lectin is prepared in PBST.)    -   Excess liquid was removed and discarded from the plate by        striking the inverted plate on paper towels.    -   Each well was washed once with PBST (using a squeeze bottle).        Excess liquid was removed and discarded by striking the inverted        plate on paper towels.

Biotinyl Lectin Binding

-   -   50 μl/well of 1 μg/ml biotinyl Erythrina cristagalli lectin in        PBST was added.    -   The plate was incubated for 1 h at room temp. (Meanwhile, the        Avidin-Biotin-Complex were prepared)    -   Excess liquid is removed and discarded by striking the inverted        plate on paper towels.    -   Each well is washed with PBST a 1^(st) time (using a squeeze        bottle). Excess liquid from the 1^(st) wash is removed by        striking inverted plate on paper towels.    -   Each well is washed with PBST a second time (using a squeeze        bottle). Excess liquid from the 2nd wash is removed by striking        inverted plate on paper towels.    -   Each well is washed with PBST a third time (using a squeeze        bottle). Excess liquid from the 3rd wash is removed by striking        inverted plate on paper towels.

Preparation of Avidin-Biotin Complex

A VECTASTAIN® ABC Reagent Kit (Vector Elite PK-6100) is used fordetection.

-   -   10 drops of REAGENT A (Avidin DH) was added to 25 ml PBST, then        10 drops of Reagent B (Biotinylated Horseradish Peroxidase) was        added.    -   Reagents were mixed and allowed to stand 30 min at room temp.

Detection of Biotinyl Lectin Bound

-   -   50 μl/well of Avidin-Biotin Complex was added.    -   The plate was left for 1 h at room temp. (Meanwhile, the ABTS        reagent is prepared.)    -   Excess liquid was removed and discarded by striking inverted        plate on paper towels.    -   Each well is washed with PBST a 1^(st) time (using a squeeze        bottle). Excess liquid from the 1^(st) wash is removed by        striking inverted plate on paper towels.    -   Each well is washed with PBST a second time (using a squeeze        bottle). Excess liquid from the 2nd wash is removed by striking        inverted plate on paper towels.    -   Each well is washed with PBST a third time (using a squeeze        bottle). Excess liquid from the 3rd wash is removed by striking        inverted plate on paper towels.    -   100 μl/well ABTS reagent (0.03% H₂O₂, 1 mM        2,2-azino-di(3-ethylbenzthiazoline) sulfonate in 0.1 M citrate,        pH 4.0). was added and the time noted.    -   The plate is incubated for exactly 30 min at room temp.    -   The plate is read A405 on Dynex Technologies MRXTC ELISA reader.

Calculations

For each plate, the median blank was subtracted from gross A405 ofstandards, controls, and unknowns. A regression line was constructed foreach plate with the 1 ng to 10 ng standards (ignoring 0.5 ng and 25 ngstandards) to use for mg/ml calculations. For each plate, mg/mlconcentrations are calculated for each unknown and control median netA405 values from triplicate wells using regression line. Median net A405values from triplicate wells were used to calculate mg/ml for eachunknown. Any excessive plate-to-plate variation in the blank, standards,or known controls was noted.

Each serum was assayed on 3 separate plates on separate days (in batchesof 4 plates). For each serum, the mg/ml results of 1st assay (sequentialorder of samples), 2nd assay (shuffled order), and 3rd assay (scrambledorder) were presented individually, along with median and the mean, SD,and SEM of the mg/ml results. Any excessive day-to-day variation inblank, standards, or known controls was noted.

TABLE 2 Typical ELISA Plate Geometry IV _1_(—) _2_(—) _3_(—) _4_(—)_5_(—) _6_(—) _7_(—) _8_(—) _9_(—) _10_(—) _11_(—) _12_(—) A PBST  10ng/ml asHP  20 ng/ml asHP 50 ng/ml asHP B PBST C8 T31 T32 PBST C N8 T33T34 D T35 T36 T37 E T38 T39 T40 F T41 T42 C5 G T43 T44 N4 H 100 ng/mlasHP 200 ng/ml asHP 500 ng/ml asHP PBST

Reagents for Assays

Rabbit anti-haptoglobin AB: Sigma catalog #H-8636.

BSA: bovine serum albumin Sigma catalog #A-3059

Biotinyl ECL: Vector catalog #B-1145

Vectastain Avidin Biotin Complex (ABC kit): Vector Elite PK-6100

PBS: (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, and 1.5 mM KH₂PO₄).10×PBS prepared inhouse.

PBST: PBS with 0.05% TWEEN-20 [Sigma Catalog #P3563]

Asialohaptoglobin standard: 500 μl 1 mg/ml haptogobin [Sigma catalog#H-3536] in water was added to 300 μl water in 15 ml tube and mixed. 200μl 0.5 NH₂SO₄ was added and mixed. Mixture was heated for 60 min at 80°C. and cooled on ice. 1 ml 10×PBS was added and mixed. 200 μl 0.5 N NaOHwas added and mixed. 8.8 ml water was added, mixed and the pH of theresulting solution checked for neutralization with pH paper. Theresulting 50 μg/ml asialohaptoglobin standard in was frozen in 200 μlaliquots at −20° C.

ABTS reagent: 0.03% H₂O₂, 1 mM 2,2-azino-di(3-ethylbenzthiazoline)sulfonate in 0.1 M citrate (pH 4.0): 30% hydrogen peroxide [Sigmacatalog #H-1009] (diluted 1000-fold for ABTS reagent); 100 mM ABTS inwater [Sigma catalog #A-1888] (store frozen in 500 μl aliquots; diluted100-fold for ABTS reagent); 1 M citrate pH 4.0 (dilute 10-fold for ABTSreagent).

Example 3 Assessment of Assay Sensitivity, Specificity andReproducibility

The high through-put ELISA assay detailed above takes advantage ofsimilarities in ligand binding between galectin-3 and the lectinErythrina cristagalli. This sandwich ELISA was used to compare 150blinded sera samples from the Early Detection Research Network (EDRN)colon reference set (normal controls, adenomas and adenocarcinomas).Results of the assays were used to constructed receiver operatingcharacteristic curves of sensitivity versus (1-specificity). The curvesshown in FIG. 3 demonstrated that the assay successfully differentiatedindividuals with colorectal neoplasia from normal controls with a highdegree of sensitivity and specificity.

The AUC for the galectin-3 (Gal3) ligand alone, normal versus cancer,was 0.84 and for Gal3 ligand+FOBT (fecal occult blood test) was 0.91(FOBT alone 0.62). The AUC for Gal3 ligand alone, normal versus allneoplasia (adenoma+carcinoma), was 0.74 and for Gal3 ligand plus FOBTnormal versus all neoplasia was 0.80. Thus, based on a newly developedassay, the serum 40-kDa haptoglobin glycoform (and galectin-3 ligand)shows promise for validation as a clinically relevant biomarker fordetection of colorectal neoplasia. The ELISA was validated within andbetween days using calibration curves and concentration reproducibility.Each unknown serum was assayed on 3 separate plates on 3 separate days.These data demonstrate that the assay is linear to 500 pg. Analyticsamples assayed on three different days were within a coefficient ofvariability of 15% (see, e.g., FIG. 4). Day to day variation in assayresults were assessed in more detail in the studies shown in FIG. 5. Asshown, while absolute quantitation of serum 40-kDa haptoglobin glycoformvaried slightly from day to day, discrimination between cancer andcontrol samples was maintained in all cases.

It was further demonstrated that assay results remained consistent overtime and when samples were stored (frozen) for extended periods of timeprior to being subjected to assay. As shown in FIG. 6, samples werequantitively assessed for the 40-kDa serum haptoglobin glycoform andthen stored for three years prior to reassessment. Despite the storagetime and the time elapsed between assays the 40-kDa glycoform levelsfrom the original assay and the reassessment correlated well. Thesestudies demonstrate not only that accurate results can be obtained fromstored frozen samples, but also that the assay system was highlyreproducible.

Example 4 High Throughput Assay Modification

In order to adapt the assay for use in a high throughput format theELISA based system was modified for use with beads. For one couplingreaction, 100 μL of uncoupled magnetic beads (MC10026-01 Bio-plex ProMagnetic COOH Beads, Region 26, 1 mL, BioRad) at the concentration of1.25×10⁷ beads/mL were put into a tube, after a vortex (Baxter S

P→Vortex Mixer) and sonicate (mini UltraAsonik™ Ney) steps. The tube waspositioned in a magnetic separator containing a strong magnet for 1 min.Then, the supernatant had to be delicately removed and 100 μL of washbuffer (PBS, 0.05% TWEEN-20, pH 7.4) were added to the uncoupled beads.The mixture was positioned in the magnetic separator for 1 min. and thesupernatant was discarded. The uncoupled beads were then resuspended in80 μL of activation buffer (0.1 M NaH₂PO₄, pH 6.2). 10 uL of fresh 50mg/mL S—NHS (Thermo Scientific, Prod #24510) in activation buffer areadded to the tube followed by the fresh addition of 10 uL of 50 mg/mLEDC (Thermo Scientific, Prod #22980). The beads were activated 20 min.at RT under agitation and in the dark. After that, activated beads werewashed twice with 150 μL of PBS with a high speed mix for 10 sec. aftereach wash. Between each wash, the supernatant was removed after 1 min.in the magnetic separator. The activated beads were resuspended with 100μL of PBS, and 9 μg of anti-haptoglobin antibody diluted in PBS wereadded into the tube. The total volume was brought to 500 uL with PBS andthe activated beads were incubated for 2 hr. at RT under agitation andin the dark. After the 2 hr. of coupling, the tube was positioned in themagnetic separator for 1 min. The supernatant was then discarded and thecoupled beads were resuspended with 500 μL of PBS. The coupled beadswere incubated 30 min. at RT, under agitation and in the dark, with 250uL of blocking buffer (PBS, 1% BSA, 0.05% Azide, pH 7.4). Finally, thetube was positioned in the magnetic separator for 1 min. and thesupernatant was removed. The coupled beads were then resuspended in 650μL of storage buffer (PBS, 0.1% BSA, 0.02% TWEEN-20, 0.05% Azide, pH7.4) and the bead concentration was estimated with a hemocytometer.

To a 96-well plate (Greiner bio-one, No. 655096), anti-haptoglobincoupled beads (2500 beads/well) were added. Then, 50 μL of haptoglobinstandards or samples were added in each well. The wells were homogenized(Mixer type 16700) and the plate was incubated 1 hr. at RT underagitation and in the dark. After that, the plate was washed twice withPBST (Bio-Plex Pro™ Wash Station). Then, 50 μL of biotinyl-ECL at theconcentration of 2.5 mg/mL prepared in staining buffer (PBS, 1%; BSA, pH7.4) were added to each well. The plate was mixed and was incubated for1 hr. at RT under agitation in the dark. The plate was then washed threetimes with PBST and finally, 50 μL of streptavidin-PE (BioRad#171-304501) at the concentration of 2 ug/mL prepared in staining bufferwere added to each well. The plate was mixed and incubated for 10 min.at RT under agitation and in the dark. Finally, 75 μL of storage bufferwas added to each well and after 2 min. of plate agitation, thestreptavidin-PE fluorescence on beads was read by a Bio-plex instrument(BioRad, Ca).

As shown in FIG. 7, assays performed on beads resulted in similarspecificity and sensitivity as compared to assays completed in wells ofmicrotiter plates (see, e.g., FIG. 3 vs. FIG. 7). Thus, these studiesdemonstrate that the assays can be replicated on the surface of beadsand are thus amenable to further commercial scale-up.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. An assay method comprising: a) diluting the serum sample of a subjectbetween 25-fold and 150,000-fold; b) desialylating the diluted serum; c)contacting the desialylated serum with an antibody that bindshaptoglobin to form a complex; and d) detecting the complex with adetectable lectin that binds galactose.
 2. The method of claim 1,wherein step (a) comprises diluting the serum at least 50-fold.
 3. Themethod of claim 1, wherein the desialylated serum is further diluted toa final dilution of between about 100-fold and 150,000-fold; about1,000-fold and 150,000-fold; or about 15,000-fold and 150,000-foldbefore step (c).
 4. The method of claim 3, wherein the desialylatedserum is further diluted to a final dilution of about 20,000-fold beforestep (c).
 5. The method of claim 1, wherein desialylating the dilutedserum comprises treating the diluted serum with a mild acid.
 6. Themethod of claim 5, wherein the mild acid is H₂SO₄.
 7. The method ofclaim 1, wherein desialylating the diluted serum comprises treating thediluted serum with a neuraminidase.
 8. The method of claim 1, whereinthe antibody that binds haptoglobin is a polyclonal antibody.
 9. Themethod of claim 1, wherein the antibody that binds haptoglobin is boundto a substrate.
 10. (canceled)
 11. The method of claim 8, wherein theantibody that binds haptoglobin is a polyclonal antibody raised againstpurified human haptoglobin.
 12. The method of claim 1, furthercomprising contacting the complex with a wash solution prior to step(d).
 13. The method of claim 12, wherein the wash solution comprises adetergent.
 14. The method of claim 1, wherein the detectable lectin ismammalian galectin-3, Ricinus communis lectin, Sophora japonica lectin,Datura stramonium lectin, Erythrina cristagalli lectin, or Lycopersiconesculentum lectin.
 15. (canceled)
 16. The method of claim 1, whereindetecting the complex with a detectable lectin, comprises contacting thecomplex with the detectable lectin and washing the resulting complexwith a wash solution prior to said detecting.
 17. The method of claim 1,wherein detecting the complex with a detectable lectin comprisesdetecting an enzymatic activity.
 18. The method of claim 17, wherein thelectin is biotinylated or comprises a conjugated enzyme.
 19. (canceled)20. The method of claim 18, wherein detecting the complex comprisescontacting the complex with an avadin-reporter conjugate.
 21. The methodof claim 20, wherein the reporter is an enzyme.
 22. An assay methodcomprising: a) desialylating a diluted serum sample from a subject, theserum sample having been diluted 25-fold and 150,000-fold; b) contactingthe desialylated serum with an antibody that binds haptoglobin to form acomplex; and c) detecting the complex with a detectable lectin thatbinds galactose. 23-26. (canceled)
 27. A kit comprising an antibody thatbinds haptoglobin and a detectable lectin that binds galactose.
 28. Amethod of treating a subject comprising performing a colonoscopy oradministering an anticancer therapy to a subject identified as a havingan elevated serum level of a complex detected by a method in accordancewith claim 22.